1. Field of the Invention
This invention relates to an illumination system for microscopy, such as that in a fluorescence microscope or a spectral microscope, and an observation or measuring method using this illumination system.
2. Description of Related Art
There are demands that microscopes should be used for high-speed observations of the behavior of molecules, the behavior of intracellular molecules, and the physiological behavior of living cells. In this case, a sample must be illuminated in such a way that light with a plurality of wavelengths is switched at a high speed.
In a conventional device, as disclosed in Japanese Patent Kokai No. Hei 9-5243, a plurality of excitation filters are mounted to a fluorescence microscope, and excitation light of different wavelengths has been switched by rotating their filter wheel.
However, with the switching technique described in Kokai No. Hie 9-5243, it is difficult to switch the wavelength at a high speed. This is attributable to problems that much time is required for changeover in excitation wavelength, the changeover of the excitation filter is required, vibration occurs, and simultaneous illumination cannot be achieved with a plurality of wavelengths.
In recent years, calcium indicators prepared on the basis of fura-2, BTC, and GFP (Green Fluorescent Protein) have been developed in particular and utilized by many researchers. As examples of these indicators, ratiometric-pericam and a pH indicator (pHluorin) are cited. Either of them is a dual wavelength excitation/single wavelength photometry type indicator.
Whenever such an indicator is used for dual wavelength excitation with single wavelength photometry, excitation light with two different wavelengths has been alternated by switching the excitation filter.
In this case, however, the problems arise that a switching speed is low, it is hard to control the balance between the intensities of excitation light with two wavelengths, and a mechanical vibration is produced when the filter is switched.
There are further demands that microscopes and caged indicators should be used to experimentally carry out high-speed observations of the behavior of molecules, the behavior of intracellular molecules, and the physiological behavior of living cells. When the concentration of calcium ions should be increased with respect to only a particular part of the cell or the concentration of a particular substance should be increased with respect to the particular part, a caged compound is used for experiment. The caged compound has the characteristic that when the compound is irradiated with ultraviolet light of wavelength 300-360 nm, it brings about a photochemical change to discharge a particular substance. An illumination system is required in which this caged reagent is loaded on the cell and the particular part of the cell is irradiated with ultraviolet rays to release the cage.
When the caged indicator is used, a costly UV laser has been required for irradiation. Alternatively, experiment has been required in which a filter wheel is mounted to a microscope to switch a filter for ultraviolet light to a filter for fluorescence observation. Since it is difficult to rotate the filter wheel at a high speed, the improvement of time resolution is impaired.
Furthermore, in the conventional microscope, as disclosed in German Patent Laid-Open Specification No. 2,626,540, a reflecting interference microscope can be used to form an image, but it is not easy to switch the illumination wavelength of reflecting interference. In the conventional microscope, it is required that the filter wheel is mounted to provide filters of different wavelengths and an image is formed in accordance with each wavelength. Moreover, the use of a plurality of costly band-pass filters involves a large expense.